Arabidopsis IWS1 Interacts with Transcription Factor BES1 and Is Involved in Plant Steroid Hormone Brassinosteroid Regulated Gene Expression

Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression

Lei Li, Huaxun Ye, Hongqing Guo, and Yanhai Yin1

Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011

Edited by Mark Estelle, The University of California San Diego, La Jolla, CA, and approved January 08, 2010 (received for review August 13, 2009) Plant steroid hormones, brassinosteroids (BRs), regulate essential knowledge about the transcriptional mechanisms by which BES1 growth and developmental processes. BRs signal throughmembrane- and BZR1 regulate gene expression is still limited. localized receptor BRI1 and several other signaling components to IWS1/SPN1 protein was firstly identified in yeast (13, 14). It was regulate the BES1 and BZR1 family transcription factors, which in turn reported to be involved in postrecruitment of RNAPII-mediated control the expression of hundreds of target genes. However, knowl- transcription and required for the expression of several inducible edge about the transcriptional mechanisms by which BES1/BZR1 genes (14). It was also copurified with RNAPII and several tran- regulate gene expression is limited. By a forward genetic approach, scription elongation factors, including Spt6, Spt4/Spt5, and TFIIS we have discovered that Arabidopsis thaliana Interact-With-Spt6 (13, 15, 16). Recent studies indicated that the yeast IWS1/SPN1 (AtIWS1), an evolutionarily conservedproteinimplicatedinRNApoly- recruited Spt6 and the SWI/SNF chromatin remodeling complex merase II (RNAPII) postrecruitment and transcriptional elongation pro- during transcriptional activation (17). Human IWS1 has been cesses, is required for BR-induced gene expression. Loss-of-function shown to facilitate mRNA export and to coordinate Spt6 and the mutations in AtIWS1 lead to overall dwarfism in Arabidopsis, reduced histone methyltransferase HYBP/Setd2 to regulate histone mod- BR response, genome-wide decrease in BR-induced gene expression, ifications (18, 19). IWS1 appears to be essential, as depletion/ and hypersensitivity to a transcription elongation inhibitor. Moreover, reduction of IWS1 gene results in lethality in yeast (14) and sig- AtIWS1 interacts with BES1 both in vitro and in vivo. Chromatin immu- nificant inhibition of human cell proliferation in vitro (20). How- noprecipitation experiments demonstrated that the presence of ever, the biological function of IWS1/SPN1 and mechanism of its AtIWS1 is enriched in transcribed as well as promoter regions of the action remain largely elusive. target genes under BR-induced conditions. Our results suggest that In this study, we found that AtIWS1, the yeast and human AtIWS1 is recruited to target genes by BES1 to promote gene expres- counterparts of which are implicated in gene expression processes sion during transcription elongation process. Recent genomic studies after RNAPII recruitment (14, 17–19, 21), interacts with tran- have indicated that a large number of genes could be regulated at steps scription factor BES1 and is required for BR-induced gene after RNAPII recruitment; however, the mechanisms for such regula- expression. Our results therefore establish an important role for tion have not been well established. The study therefore not only AtIWS1 in plant steroid–induced gene expression and provide establishes an important role for AtIWS1 in plant steroid–induced gene insights into the functions of this conserved protein across eukar- expression but also suggests an exciting possibility that IWS1 protein yotes as well as into the mechanism of transcriptional regulation. can function as a target for pathway-specific activators, thereby providing a unique mechanism for the control of gene expression. Results seb1 Suppresses bes1-D Phenotype and Has Reduced BR Response. In plant growth | transcription regulation | transcription elongation | RNA an attempt to identify BES1 downstream signaling components, polymerase II | Spt6 we screened for suppressors of bes1-D gain-of-function mutant. One of the suppressors, bes1-D seb1, was selected for further lant steroid hormones, brassinosteroids (BRs), regulate studies because of its strong suppression of the bes1-D phenotype Pdiverse plant growth and developmental processes such as cell (Fig. 1). seb1 is a single recessive mutation, as the F1 seedlings of elongation, vascular development, photomorphogenesis, repro- bes1-D seb1 and bes1-D cross displayed bes1-D phenotype and F2 duction, and stress responses (1, 2). Unlike animal steroid hor- segregated suppressors showed a 3:1 ratio. Whereas bes1-D mones that bind to nuclear receptors to directly regulate expression seedlings displayed excessive stem elongation, bes1-D seb1 of target genes, BRs function through membrane-localized recep- seedlings had reduced stem elongation (Fig. 1A). Unlike bes1-D, tor BRI1 and several other components to regulate the protein which was resistant to BRZ inhibition in hypocotyl elongation levels and activities of BES1 and BZR1 family transcription factors assays, the bes1-D seb1 double mutant restored BRZ sensitivity, (3–8), which have six members in Arabidopsis. Although knockout suggesting that this suppressor gene is required for the bes1-D of BES1 alone does not give obvious phenotype, knockdown of phenotype (Fig. 1B). BES1 and BZR1 by RNAi leads to a semidwarf phenotype, sug- We next cloned the SEB1 gene by map based cloning (Fig. gesting that this family of proteins function redundantly in BR S1A). The seb1 mutation was first narrowed down to a 60-kb signaling (9). BES1 and BZR1 have atypical basic helix–loop–helix region by a set of flanking markers. Five candidate genes in the (bHLH) DNA binding domain and bind to E-box (CANNTG) and/ interval were then sequenced. A “G” to “A” mutation in Ara- or BRRE (BR Response Element, CGTGT/CG) to regulate BR target gene expression (9, 10). In addition, BES1 interacts with other transcription factors such as BIM1 and AtMYB30 as well as Author contributions: L.L. and Y.Y. designed research; L.L. performed research; L.L. and H.Y. putative histone demethylases ELF6 and REF6 (9, 11, 12). Con- contributed new reagents/analytic tools; L.L., H.Y., and M.G. analyzed data; and L.L., M.G., sistent with the important roles of BES1 in mediating BR signaling, and Y.Y. wrote the paper. the gain-of-function mutant bes1-D, in which BES1 protein accu- The authors declare no conflict of interest. mulates to high level, displays constitutive BR response pheno- This article is a PNAS Direct Submission. types, including excessive cell elongation, resistance to the BR 1To whom correspondence should be addressed. E-mail: [email protected]. biosynthesis inhibitor brassinazole (BRZ), and increased expres- This article contains supporting information online at www.pnas.org/cgi/content/full/ sion of BR-induced genes (7). Despite the emerging findings, 0909198107/DCSupplemental.

3918–3923 | PNAS | February 23, 2010 | vol. 107 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.0909198107 Downloaded by guest on October 3, 2021 BR-Induced Gene Expression Is Decreased in seb1 Mutant. To understand the molecular basis for impaired BR response and growth phenotype of the seb1 mutant, we first tested the expression of several known BR target genes using quantitative RT- PCR. The expression of all four genes tested were significantly reduced in the seb1 mutant and increased in the bes1-D mutant, especially under BL-induced conditions (Fig. 2A). To gain a global view of how many BR-regulated genes are affected in the mutants, we performed microarray analyses using WT, seb1, and bes1-D seedlings (all in En-2 background) with or without BL treatments. These experiments identified 3138 and 3824 differentially expressed genes in seb1 and bes1-D, respectively, compared with WT (Fig. S5). The substantial overlap (1437 genes) between AtIWS1- and BES1-regulated genes suggests that AtIWS1 plays important roles in the BR signaling pathway. To understand this further, we analyzed in greater depth the genes that were induced by BL and were reduced in seb1 mutant (Fig. 2 B and C). Of the 406 genes that were induced by BL, ≈70% were Fig. 1. seb1 Suppresses bes1-D phenotype and has reduced BR response. (A) up-regulated in bes1-D (Fig. 2C and Table S1). Comparison of the Two-week-old light-grown seedlings. (B) Hypocotyl elongation assay with 2- seb1 and WT gene expression profiles indicated that a large week-old light-grown seedlings in the absence or presence of 1000 nM BRZ. fraction of the BL-responsive genes were also regulated by (C) Two-week-old light-growing seedlings of Col-0, T-DNA insertion line AtIWS1: 31% (127/406) were reduced in seb1 in the presence of atiws1 (SALK_056238) in Col-0 background, En-2, and seb1 single mutant in BL, whereas 16% (66 / 406) were reduced in seb1 in the absence of En-2 background. (D) Hypocotyl elongation assays with 2-week-old light- B – grown seedlings (0 and 100 nM BL), or 5-day-old dark-growing seedlings (0 BL (Fig. 2 and Table S1 Table S3). Most of the BL-induced or 1,000 nM BRZ). A total of 15–20 seedlings were used for each line for each genes that were reduced in seb1 (59/66) were also included in the condition and the difference between mutants and wild-type were sig- 127 genes reduced in seb1 in the presence of BL (Fig. 2B). The nificant, as measured by Student’s t test (P < 0.05). same conclusion can be drawn from the gene clustering analysis with the 406 BR-induced genes (Fig. 2C). These genome-wide analyses revealed that AtIWS1 plays important roles in BL- bidopsis fi gene At1g32130 was identi ed, which created an early stop induced gene expression. codon in the middle of the gene (Fig. S1B). Expression of a genomic To further understand the reduced growth phenotype of seb1, clone of At1g32130 in the bes1-D seb1 mutant complemented the we examined genes implicated in cell elongation (Table 1). BRs dwarf phenotype (Fig. S1C), indicating that the identified mutation promote cell elongation by inducing the expression of the genes is responsible for the seb1 phenotype. At1g32130 and its close homolog At4g19000 (44% protein sequence identity) have not previously been characterized in plants, and the C-terminal domain shows high homology to C-termini of IWS1 proteins found in other species, including yeast and human beings (Fig. S1D). We therefore named these two Arabidopsis genes AtIWS1 and AtIWS2, respectively. The seb1 mutant by itself also shows a dwarf phenotype compared to En-2 wild-type (Fig. 1C). In addition, we obtained a mutant line, atiws1, which contains a T-DNA insertion in the third

exon of the AtIWS1 gene, most likely resulting in a null allele (Fig. PLANT BIOLOGY S2). The atiws1 in Col-0 background and the seb1 in En-2 background, both null alleles, have similar semidwarf phenotypes with reduced stem elongation in both seedling and adult plants (Fig. 1C and Fig. S3). Whereas AtIWS1 has high levels of expression in both seedling and adult plants, AtIWS2 has barely detectable expression in general (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Consistent with the gene expression data, the T-DNA knockout line atiws2 shows no visible growth phenotype (Fig. S4). Furthermore, the double knockout line atiws1atiws2 and seb1atiws2 do not enhance the atiws1/seb1 semidwarf phenotype (Fig. S4). We therefore conclude that AtIWS1 is the active member in this gene family in BR signaling. The seb1 and atiws1 mutants not only have overall dwarf phenotypes, but also exhibit altered BR responses in the hypocotyl D elongation assays (Fig. 1 ). Under light, the hypocotyl lengths of Fig. 2. BR induced gene expression is decreased in seb1 mutant. (A) the seb1 and atiws1 mutants were slightly shorter than the wild-type Quantitative RT-PCR was performed with 2-week-old seedlings treated with (WT) controls in the absence of BL; however, BL-induced hypo- or without 1,000 nM BL. The gene expression levels were signiﬁcantly cotyl elongation was greatly reduced in both mutants compared reduced in seb1 and increased in bes1-D as tested by two-way ANOVA F test with WT. Similarly, in the dark, whereas mutant seedlings were (P < 0.01). The average and standard deviations were from three biological ∼20% shorter than WT without BRZ treatment, they were 50% repeats. (B) Venn diagram shows the overlap genes among different groups. μ The lists of BL-induced genes and those reduced in seb1 with or without BL shorter when treated with 1 M BRZ, suggesting that the mutants treatments are presented in Table S1, Table S2, and Table S3 (C) Clustering are hypersensitive to reduced BR levels. These results demon- analysis of 406 BL-induced genes by GeneSpring program. Yellow rectangles strated that AtIWS1 is required for BR-induced cell elongation. indicated genes reduced in seb1 in the presence of BL.

Li et al. PNAS | February 23, 2010 | vol. 107 | no. 8 | 3919 Downloaded by guest on October 3, 2021 Table 1. Expression levels of cell elongation related genes in seb1 and bes1-D mutants En-2 seb1 bes1-D Gene Gene no. Mock SD BL SD Mock SD BL SD Mock SD BL SD name

AT1G10550 356 46 854 58 207 5 396 13 1,444 100 1,490 39 XTH33 AT4G30290 468 127 3,210 150 537 22 2,107 249 4,389 22 9,575 322 XTH19 AT1G65310 365 13 1,298 102 431 73 912 67 1,671 73 2,335 535 XTH17 AT3G07010 423 43 975 40 340 40 647 77 800 40 1,100 66 Pectate lyase AT3G23730 1,365 205 3,087 170 996 36 1,670 93 1,570 36 3,138 24 XTR7 AT4G25810 896 132 1,836 425 497 87 889 153 4,951 87 7,486 893 XTR6 AT5G57560 166 25 273 26 142 22 196 4 305 22 333 62 XTH22/TCH4 AT1G04680 1,012 131 1,657 90 676 65 938 89 813 65 1,164 199 Pectate lyase AT3G29030 381 32 551 63 225 34 302 26 549 34 420 59 ATEXPA5 AT2G37640 1,630 226 2,242 194 759 101 1,124 60 1,839 101 1,681 25 ATEXPA3 AT3G10720 2,237 201 3,030 288 2,828 115 2,127 53 5,244 115 4,917 134 Pectinesterase

Gene expression levels were determined by microarray analysis with GeneChip Affymetrix Arabidopsis ATH1 Genome Arrays. Averages and SDs are derived from three biological repeats.

involved in cell wall–loosening or -modifying enzymes, including sion, coexpression of AtIWS1 and BES1 significantly activated xyloglucan endotransglycosylase/hydrolase (XTH), xyloglucan the reporter gene expression by both qualitative (Fig. 3A) and endotransglycosylase–related (XTR), pectin lyase, pectinester- quantitative (Fig. 3B) assays. We also made a BES1-C-terminal ase, and expansins (ATEXP) (22–24). There are 27 cell wall– deletion mutant that did not show activation by itself; however, related genes that are induced by BL (Table S1); 11 of these genes coexpression with AtIWS1 still led to strong activation of the have reduced expression in seb1 and up-regulated expression in reporter gene, although AtIWS1 itself did not activate the bes1-D (Table 1). In most of the cases, the induction by BL is also reporter gene expression (Fig. 3 A and B). This observation reduced in seb1 compared with WT. Taken together, the global indicated that BES1 and AtIWS1 interacted with each other in gene expression studies indicated that the expression of many BL- yeast. We next mapped the interaction site on BES1 by GST induced genes, including those involved in cell elongation, is pull-down assays and found that the central part of BES1 (aa reduced in seb1 mutant, which explains the impaired growth and 140–271) was sufficient for interaction with AtIWS1 (Fig. 3C). BR response phenotypes of the mutants. We also tested whether BES1 interacts with AtIWS1 in vivo by Bimolecular Fluorescence Complementation (BiFC) experiments AtIWS1 Interacts with BES1 Both in Vitro and in Vivo. The sup- (25) using an Arabidopsis protoplast expression system (26). BES1 pression of bes1-D phenotype by seb1 and the reduction of BL- and AtIWS1 were fused to C-terminal YFP and N-terminal YFP, induced gene expression in the mutant prompted us to test the respectively. Strong fluorescence can be observed in the nucleus hypothesis that AtIWS1 directly interacted with BES1. We first when BES1-cYFP and AtIWS1-nYFP were cotransformed into tested the interaction using a yeast–two-hybrid system. Although the protoplasts, indicating the in vivo interaction of BES1 with BES1 by itself moderately increased the reporter gene expres- AtIWS1 proteins (Fig. 3D). No positive signals were observed

Fig. 3. AtIWS1 interacts with BES1 both in vitro and in vivo. In yeast two-hybrid experiments to detect direct protein–protein interactions, β-galactosidase (LacZ) activity was detected with X-gal (A) or with ONPG in a quantitative liquid-culture assay (B). (C) GST pull-down experiments using MBP-AtIWS1 and GST tagged full-length or truncated BES1. Full-length BES1 includes a DNA binding domain (DNA BD), BIN2 phosphorylation domain (Phospho), PEST motif, and the C-terminal domain. AtIWS1 was detected by a Western blot with anti-MBP antibody. (D) Cotransformation of Arabidopsis protoplasts with BES1-cYFP and AtIWS1-nYFP lead to the reconstitution of YFP activity in the nuclei, as indicated by arrows. (E) Cotransformation of cYFP and AtIWS1-nYFP did not produce any positive signals.

3920 | www.pnas.org/cgi/doi/10.1073/pnas.0909198107 Li et al. Downloaded by guest on October 3, 2021 when AtIWS1-nYFP was coexpressed with cYFP (Fig. 3E). Con- strong association with the transcribed regions and moderate sistent with its interaction with BES1, AtIWS1-GFP is localized in association with the promoters, only in the presence of BL (Fig. 4D). the nucleus (Fig. S6). This demonstrated a correlation between the activation of BR signaling/BES1 accumulation and the association of AtIWS1 to the AtIWS1 Is Likely Involved in Transcription Elongation. In yeast and target genes. These results are consistent with the notion that IWS1 human systems, IWS1/SPN1 is an important transcriptional reg- functions at postrecruitment and transcription elongation steps. ulator with multiple critical roles that are not fully understood. It is implicated in transcription elongation by interacting directly Discussion with elongation factor Spt6. We reasoned that if AtIWS1 were In this study, we found that loss of function in SEB1/AtIWS1 gene functionally similar to IWS1 from yeast and animal systems, it significantly reduced BR response as measured by hypocotyl elon- would interact with the conserved Spt6 (At1g65440) in Arabi- gation assay and caused genome-wide decrease in BR target gene dopsis as well. We then tested the hypothesis and found that the expression. AtIWS1 was found to directly interact with BES1 by conserved N terminus of AtSpt6 (1–482aa) interacts with either yeast–two-hybrid and bimolecular fluorescence complementation full-length or C terminus (203–502aa) of AtIWS1 in the yeast– assays as well as GST pull-down experiment. AtIWS1 interacted two-hybrid assay (Fig. 4A). with transcription elongation factor Spt6 through conserved The association of AtIWS1 with AtSpt6 implies that AtIWS1 is domains in each of the proteins. Moreover, loss-of-function mutant IWS1 seb1 involved in transcription elongation, like IWS1/SPN1 in other of gene showed hypersensitive response to transcription systems. To test the hypothesis, we examined the germination and elongation inhibitor 6-AU. Finally, the ChIP experiments demon- growth of seb1 in medium containing 6-azauracil (6-AU), which is strated that AtIWS1 binding was enriched at both transcribed and commonly used as transcription elongation inhibitor in yeast since promoter regions in the target genes, which was clearly different it can cause depletion of free nucleotide levels therefore decrease from that of BES1, which is enriched only in promoter regions. transcription elongation efficiency (27). As the concentration of Based on these data, we propose a model in which BES1 binds directly to AtIWS1 and recruits it to BR-responsive gene promoters 6-AU was increased, both the germination rate and plant growth E were inhibited. More significantly, seb1 was more sensitive to the under BR-induced conditions (Fig. 4 ). This model predicts that the seb1 phenotype would be more evident when the BR pathway inhibitor (Fig. 4B), supporting a role for AtIWS1 in transcription was activated, which was proved by both the BR response and the elongation. genome-wide gene expression analyses of the seb1 mutant. If AtIWS1 is indeed involved in RNAPII postrecruitment and All of the available evidence suggested that IWS1 family proteins transcription elongation steps in Arabidopsis, the protein should be function after RNAPII recruitment steps to regulate gene expres- associated with the transcribed regions of the target genes in vivo, ’ sion, although the molecular mechanisms of IWS1 action remain to which should differ from the BES1 s binding patterns. We used be fully defined. Yeast IWS1/SPN1 was originally identified in a Chromatin immunoprecipitation (ChIP)-qPCR to test the hypoth- genetic screen with a mutation that suppresses a postrecruitment- esis. Two BES1 target genes (At1g64380 and At2g22810/ACS4) defective TBP (TATA binding protein) allele (14). Careful genetic were chosen because of their long promoters and ORFs that would studies indicated that IWS1/SPN1 functions at the postrecruitment facilitate the ChIP assays (Fig. S7). As expected, BES1 was shown to step (14). IWS1 was also implicated in transcription elongation, as interact mostly with the promoters but not the transcribed regionsor it interacts with transcription elongation factor and histone chap- ′ C the 3 regions in both genes (Fig. 4 ). In contrast, AtIWS1 showed erone Spt6 and was copurified with several transcription elongation complexes (13, 15, 16). In this study, we showed several lines of evidence that, like its counterparts in yeast and animal systems, AtIWS1 is also involved in RNAPII postrecruitment/transcription elongation processes. Our study provides compelling evidence that regulation of gene expression can occur after RNAPII recruitment. Transcription is a multiphase, highly concerted process, involving numerous factors PLANT BIOLOGY at many stages. Although previous studies of transcription regulation were primarily focused on the initiation step, the importance of the regulation at transcription elongation has started to be recognized more recently (28–32). Recent genomic studies suggest that a large portion of genes in a mammalian system can be regulated at postinitiation steps (29, 31). For example, it has been shown that hundreds of human genes had no detectable transcripts while their promoters were occupied by transcription preinitiation complex (31). Similarly, it was also observed in human cells that more than 30% of genes had active histone modification markers as well as initiating form of RNAPII at their promoters without detectable transcripts (29). The widespread phenomena may be explained by various mechanisms including abortive initiation, low processivity, and promoter-proximal pausing of RNAP II (29). Yet Fig. 4. AtIWS1 is likely involved in transcription elongation. (A) Yeast-two- the molecular mechanisms by which the large number of genes is hybrid assays to detect interactions between AtIWS1 or AtIWS1 C-terminal regulated after RNAPII recruitment by cellular signaling remain (IWS1 domain) and Arabidopsis Spt6 (At1g65440) N-terminal domain. (B) to be fully understood. Some transcriptional activators can influence Four-week-old plants in the media with indicated concentrations (mg/L) of subsequent steps including promoter clearance (33), promoter- 6-AU. The germination rates were determined after 3-week of culture under proximal pausing through P-TEFb (34), and elongation rate of light. The assay was repeated three times, and representative results are shown. (C) ChIP using bes1-D seedlings with anti-BES1 antibody and control RNAPII possibly by interacting with TFIIH (35). For example, the IgG. (D) ChIP using AtIWS1-TAP transgenic and WT seedlings. The 5srRNA heat shock activator HSF can indirectly recruit P-TEFb complex, was used as internal control for C and D. The average and standard devia- which phosphorylates RNAP II and stimulates promoter-paused tions were derived from three to four biological replicates. (E) A model for RNAPII to enter into productive elongation in response to heat AtIWS1 function in BES1 mediated gene expression. shock (36). In addition, certain types of transcription activators

Li et al. PNAS | February 23, 2010 | vol. 107 | no. 8 | 3921 Downloaded by guest on October 3, 2021 were able to promote transcription elongation in vitro, correlating Materials and Methods with their abilities to interact with TFIIH, the kinase activity of Plant Materials, Growth Conditions, and Hypocotyl Elongation Assays. Arabi- which can convert RNA polymerase from a nonprocessive to a dopsis thaliana ecotype Columbia (Col-0) and Enkheim-2 (En-2) were used as processive form (32, 35, 37). The genetic, genomic, and molecular wild-type controls. Plant transformation, growth conditions, and hypocotyl evidence presented in this study clearly demonstrates that the elongation assays were carried out as previously described (11). The AtIWS1 transcriptional factor BES1 can recruit AtIWS1 to promote plant and AtIWS2 T-DNA knockout lines are SALK_056238 and SALK_006346, respectively (39). steroid hormone–regulated gene expression. Our study provides genetic evidence that a transcription regulator involved in post- bes1-D Suppressor Screen. Approximately 40,000 bes1-D seeds were treated recruitment/transcription elongation can be directly targeted by a with 0.2% EMS (ehyl methanesulfonate) for 14 h and grown in 40 pools pathway-specific transcriptional factor, which reveals a unique (1,000/pool). M2 seeds from each pool were grown on 1/2MS medium that regulatory mechanism for the control of gene expression. contains 1 μM Brassinazole (a gift from Prof. Tadao Asami) under light. Our results extended our understanding about the mechanisms Although bes1-D seedlings were resistant to the inhibitory effect of BRZ, of BES1-regulated gene expression. We previously showed that suppressors that showed sensitivity to the inhibitor were selected. The fi BES1 could interact with ELF6 (Early Flowering 6) and its homolog putative suppressors were con rmed in M3 generation and backcrossed to bes1-D to determine the dominant/recessive nature of the mutations. REF6 (Relative of Early Flowering 6), which are Jumonji N/C (JmjN/C) domain-containing histone demethylases, to modulate fl Map-Based Cloning. The bes1-D seb1 double mutant in En-2 background was BR responses and other developmental processes such as owering crossed to bes1-D single mutant in polymorphic Wassilewskija (Ws) back- time control. The study of AtIWS1, on the other hand, shows that ground. All of the F1 offspring showed bes1-D phenotype, indicating that BES1 not only participates in histone modifications during tran- seb1 is a recessive mutation. In the F2 generation, approximately three scription initiation but can also regulate postrecruitment/tran- fourths of plants showed bes1-D phenotype, whereas the remaining one scription elongation by interacting with factors such as AtIWS1. It fourth of plants showed bes1-D seb1 phenotype, suggesting that seb1 is a has been reported that TRIP-1 (TGF-beta Receptor Interacting single gene mutation. Those bes1-D seb1 plants in F2 generation were used as mapping population. The mutation was narrowed down to 60 kb on Protein-1), an essential subunit of eIF3 protein translation initiation chromosome 1 by using ∼800 bes1-D seb1 plants and 10 CAPS (40), dCAPS complex, is phosphorylated by BR receptor BRI1, which may (41), or SSLP (42) markers (M1–M10; Table S4). Five candidate genes in this modulate its activity and influence protein translation (38). BR interval were then sequenced, and a single base pair change was detected. signaling, therefore, can affect the expression of target genes at multiple levels, including chromatin modification and transcription Gene Expression and Microarray Analysis. Total RNA was extracted and initiation/elongation, as well as translation. purified from seedlings of different genotypes and treatments using RNeasy Our results provide important insights into the biological func- Mini Kit (Qiagen) with on-column DNase digestion, following the manu- ’ tion of IWS1 family proteins. Despite its apparent importance in facturers instructions. Microarray experiments were performed using Affy- the regulation of gene expression, the biological functions of IWS1 metrix ATH1 Genome Arrays with 2-week-old light grown En2, seb1 and bes1-D seedlings with long-day (16 h) conditions. The plants were treated family proteins remain elusive. Yeast IWS1 is essential as complete with either 1 μM BL or DMSO as control in liquid 1/2 MS medium for 3 h. loss-of-function mutant is lethal (14). Similarly, reduction of human Triplicate samples were collected and processed for RNA extraction. Probe IWS1 expression by RNAi leads to the arrest of cell growth (20). labeling, hybridization, and scanning were performed in GeneChip facility at However, a nonlethal allele of yeast IWS1/SPN1 reveals that IWS1/ Iowa State University according to the manufacturer’s instructions. Micro- SPN1 is required for the inducible expression of several genes, array data were normalized by R using MAS 5.0 method in the affy package, including PHO5 induced by phosphate starvation as well as both and the log2 transformed data set was then analyzed using the limma HIS3 and HIS4 by histidine deprivation (14). Similarly, we have package. The false discovery rate (FDR) was controlled at 5% using the Benjamini–Hochberg method, and genes with adjusted P value <0.05 were observed that AtIWS1 was required for BL induction of many BR considered to be significantly differentially expressed (43). The clustering target genes, including those required for cell elongation (Table 1). analysis was done with GeneSpring program from Agilent Technologies. Consistent with the reduced expression of the BR-induced genes, Quantitative RT-PCR was performed as previously described (11). The 5srRNA atiws1/seb1 mutant displayed defect in responding to BRs and was used as reference gene. Three biological replicates and 2–3 technical overall stunted growth phenotype. Therefore, IWS1 may function replicates for each biological replicate were used. to respond to various environmental conditions to regulate gene expression for optimal growth and fitness. In addition to affecting Plasmid Construction. All primers used in this study are listed in Table S5 For approximately one third of BR-regulated genes, AtIWS1 appears recombinant protein and GST pull-down assay, AtIWS1 coding region was amplified from Col-0 cDNA and incorporated into the pETMALc-H vector (44). to influence up to 14% of all genes genome-wide (Fig. 3B and Fig. seb1 BES1 and various deletion constructs were cloned into pET42a(+) (Novagen). For S5). Although some of the differential gene expression in transgenic GFP plants, AtIWS1 ORF was fused with GFP tag flanked by BRI1 mutant may be due to the pleiotropic effect, it is very likely that promoter (45) and RBCS terminator in pPZP211 (46). For transgenic AtIWS1-TAP IWS1 also regulates gene expression in other processes, in addi- (Tandem Affinity Purification)-tag plants, AtIWS1 ORF was fused into vector tion to the established role in BR-regulated gene expression. p35SCTAP with 35S-promoter and the tag containing a protein A module, a six Unlike in yeast and human systems (14, 20), AtIWS1 is not His repeat (6× His) and nine copies of a myc repeat (9xmyc) (47). For bimolecular fl essential for Arabidopsis survival. Although Arabidopsis seems to uorescence complementation assay, the constructs of N or C -terminus of EYFP were as described previously (12). The coding region of AtIWS1 and BES1 were be the only species with two copies of IWS1 genes among the inserted upstream of YFP-N or YFP-C constructs, respectively. model organisms, the two genes do not seem to function redundantly (Fig. S4). The difference in phenotypic severity between Phylogenetic Analysis. Protein sequences were obtained from NCBI, and Arabidopsis and other systems may be due to the possibility that aligned by ClustalX 2.0 (http://bips.u-strasbg.fr/fr/Documentation/ClustalX). the target genes of IWS1 are essential in yeast and human beings Mrbayes program (http://mrbayes.csit.fsu.edu/index.php) was used to recon- but not in plants, or may be due to the existence of possible struct the evolutionary tree. After 500,000 iterations in Mrbayes, the split functional redundant counterparts in Arabidopsis. Nevertheless, frequency reached as low as 0.005, which suggested high confidence of con- the viable seb1/atiws1 mutant provides a unique tool to dissect the vergence. The tree was viewed by TreeViewX (http://darwin.zoology.gla.ac. uk/_rpage/treeviewx). functions of IWS1 family proteins by genetic and genomic approaches. Further studies should help to reveal the molecular ChIP Assays. Chromatin immunoprecipitation (ChIP) was performed as functions of this important and conserved protein family in gene described by Craig Pikaard lab based on published methods (48, 49) (http:// expression and their biological roles in growth and development www.biology.wustl.edu/pikaard/) with 2-week-old bes1-D seedlings using in responding to environmental cues. antibodies against BES1 or IgG (Millipore) control for immunoprecipitation.

3922 | www.pnas.org/cgi/doi/10.1073/pnas.0909198107 Li et al. Downloaded by guest on October 3, 2021 The method for ChIP with 2-week-old WT and AtIWS1-TAP seedlings was fluorescence microscope with an YFP filter, 16–24 h after transfection. The slightly modified, using IgG Sepharose beads for immunoprecipitation assay was repeated two times with different positive rates (5–10%), depend- instead of antibody and protein A beads. The ChIP experiments were per- ing on transfection efficiency. formed 3–4 independent times. Yeast Two-Hybrid and lacZ Assays. Yeast two-hybrid assay was done as GST Pull-Down Assay. The assay was performed as described previously (7). described by Yeast Protocols Handbook (Clontech). Yeast (Y187) transformed fi BES1 and its fragments fused with GST (GST) were puri ed with glutathione with indicated constructs was grown in media lacking Trp and Leu. Positive beads (Sigma). AtIWS1 fused with Maltose Binding Protein (MBP) was puri- clones were used for LacZ assay using X-gal (5-bromo-4-chloro-3-indolyl-b-D- fi μ ed with Amylose resin (NEB). Approximately 5 g proteins were used in galactopyranoside; Sigma) or quantitative liquid assay using ONPG (ortho- each assay. The products then were detected by Western Blotting using nitrophenyl-b-D-galactopyranoside; Sigma). antibody against MBP tag (NEB). Pull-down assays were repeated three times with similar results. ACKNOWLEDGMENTS. We thank ABRC for Arabidopsis T-DNA mutants, Tadao Asami (University of Tokyo) for providing BRZ, Bing Yang for vector Bimolecular Fluorescence Complementation. Arabidopsis mesophyll proto- p35SCTAP, and Jo Anne Powell-Coffman for critical comments on the plasts were prepared and transformed by PEG-mediated transfection as manuscripts. This work was supported by National Science Foundation Grant described previously (26). Protoplasts were observed under an Olympus IX71 IOS-0546503 and a faculty start-up fund from Iowa State University (to Y.Y.).

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